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Pathogens in the genus Campylobacter are the most common cause of food-borne bacterial gastro-enteritis. Campylobacteriosis, caused principally by Campylobacter jejuni and Campylobacter coli, is transmitted to humans by food of animal origin, especially poultry. As for many pathogens, antimicrobial resistance in Campylobacter is increasing at an alarming rate. Erythromycin prescription is the treatment of choice for clinical cases requiring antimicrobial therapy but this is compromised by mobility of the erythromycin resistance gene erm(B) between strains. Here, we evaluate resistance to six antimicrobials in 170 Campylobacter isolates (133 C. coli and 37 C. jejuni) from turkeys. Erythromycin resistant isolates (n = 85; 81 C. coli and 4 C. jejuni) were screened for the presence of the erm(B) gene, that has not previously been identified in isolates from turkeys. The genomes of two positive C. coli isolates were sequenced and in both isolates the erm(B) gene clustered with resistance determinants against aminoglycosides plus tetracycline, including aad9, aadE, aph(2″)-IIIa, aph(3′)-IIIa, and tet(O) genes. Comparative genomic analysis identified identical erm(B) sequences among Campylobacter from turkeys, Streptococcus suis from pigs and Enterococcus faecium and Clostridium difficile from humans. This is consistent with multiple horizontal transfer events among different bacterial species colonizing turkeys. This example highlights the potential for dissemination of antimicrobial resistance across bacterial species boundaries which may compromise their effectiveness in antimicrobial therapy.

Improved understanding of the distribution of resistance genes within bacterial species in different host niches, and the mobility of these genes between populations, could be important for identifying source and sink populations. In the case of Campylobacter, the erm(B) gene has been identified in C. coli isolates from chicken, ducks, swine, and humans and from C. jejuni isolated from chicken (Qin et al., 2014; Wang et al., 2014; Deng et al., 2015) but other host species may be relevant. Turkeys are among the top 10 farmed animals in Europe and the United States with an estimated 323 million birds reared anually (FAOSTAT, 2017). While studies have shown that turkeys are an important host species harboring large numbers of C. jejuni and C. coli, the resistance status of these strains is not well-characterized. This has lead to the inclusion of this animal species in international surveillance programs to evaluate the levels of antibiotic resistance. In this study we carried out combined molecular microbiology and whole genome sequencing approaches to evaluate the presence of erm(B) and it’s genetic background in Campylobacter isolates obtained from turkeys sampled in Spain. The comparison with erm(B) sequences from other host species might allow further description of the microevolutionary events associated with the acquisition of this antibiotic resistance genes in Campylobacter.

Materials and Methods

Strains and Growth Conditions

Campylobacter isolates were recovered in 2014 (n = 170; 133 C. coli and 37 C. jejuni) from turkey samples obtained in the framework of the European Antimicrobial Resistance Surveillance program (DC 652/2013) in Spain (European Comission, 2013). Samples were collected at the largest turkey slaughterhouses in Spain located in different regions within the country. Each Campylobacter isolate represented a single farm and they were obtained by culturing pooled feces from turkeys (117 pooled samples: 10 animals per pool, 1170 individual fecal samples analyzed). Each pooled sample was cultured on Campylobacter blood-free selective medium (CCDA) (Oxoid). Inoculated media were incubated at 42°C for 48 h under microaerobic conditions with a commercial gas-generating system (atmosphere generator system, Oxoid). Suspected colonies were subcultured onto blood agar (BioMérieux) at 37°C for 48 h. All strains were identified by conventional multiplex PCR of the genus Campylobacter that allows the differentiation between C. coli and C. jejuni with specific primers, as described previously (Ugarte-Ruiz et al., 2012).

Antimicrobial Susceptibility Testing

Broth microdilution methods were performed to determine the antimicrobial susceptibility of the Campylobacter isolates [minimum inhibitory concentrations (MICs)]. The following antimicrobials were tested: tetracycline, ciprofloxacin, nalidixic acid, erythromycin, streptomycin, and gentamicin. Isolates were grown on blood agar plates (bioMérieux) and incubated for 48 h at 37°C under microaerophilic conditions. Growth from these cultures was suspended in sterilized water and adjusted at 0.5 McFarland. Fifty microliters of these inocula were added to 11 mL of cation-adjusted Mueller-Hinton broth (TREK Diagnostics Systems), and supplemented with 600 μL of lysed horse blood prepared in house from defibrinated horse blood (Oxoid). EUCAMP2 microdilution plates (TREK Diagnostics Systems) were inoculated and incubated under microaerophilic conditions at 37°C for 48 h. C. jejuni strain ATCC 33560 was used as a control for antimicrobial susceptibility test. Following the commission decision 2013/652/UE, the epidemiological cut-offs values considered were those described by EUCAST (EUCAST, 2017). Campylobacter isolates resistant to erythromycin (MICs: >8 mg/L to C. coli and >4 mg/L to C. jejuni) were selected to evaluate the presence of erm(B) gene.

Identification of erm(B) Gene and Whole-Genome Sequencing

The RNA methylase gene erm(B) was identified by PCR as described previously (Chen et al., 2007). Amplicons were detected by gel electrophoresis using 2% agarose gels containing 10 mg/ml SYBR Safe DNA gel stain (Invitrogen) for 40 min at 100 mA. DNA fractions were sequenced and compared using Sanger sequencing and MEGA software (version 5.05) respectively (Tamura et al., 2011; Heather and Chain, 2016). erm(B)-positive Campylobacter isolates were selected for whole genome sequencing. For DNA extraction, Campylobacter isolates were grown on blood agar plates (48 h at 42°C under microaerophilic conditions) and DNA was extracted using a QiAmp DNA mini kit (Qiagen). DNA was quantified using a Nanodrop spectrophotometer before sequencing. High-throughput genome sequencing was performed using a benchtop MiSeq sequencer (Illumina), and the short read paired-end data was assembled using the de novo assembly algorithm, SPAdes (Bankevich et al., 2012). Genome sequences were archived in the web-accessible Bacterial Isolate Genome Sequence Database: BIGSdb (Jolley and Maiden, 2010), which included functionality for identifying MLST profiles based on the pubMLST database1. Allelic diversity was evaluated using a gene-by-gene approach for genome alignment and comparison with the BLAST algorithm as previously described (Sheppard et al., 2012).

Results

Antimicrobial Susceptibility Testing

A total of 170 Campylobacter isolates (133 C. coli and 37 C. jejuni) were tested for susceptibility to six antimicrobials. Antimicrobial resistance profiles (Table 1) and MIC distributions were recorded (Supplementary Table S1). The highest proportion of antimicrobial resistance was to tetracycline (168/170; 98.8%) followed by nalidixic acid/ciprofloxacin (164/170; 96.4%), erythromycin (85/170; 50%), streptomycin (82/170; 48.2%), and finally gentamicin (13/170; 7.6%). Considering C. coli and C. jejuni separately, higher prevalence of resistance was observed in C. coli for erythromycin, streptomycin, and gentamicin (Fisher’s exact test: p < 0.001). Based upon The European Food Safety Authority (EFSA) criteria for quantifying multi-drug resistance (MDR; resistance to at least three classes of antimicrobials tested), seven MDR profiles were recorded for Campylobacter isolates (Table 1) (European Food Safety Authority [EFSA]/European Centre for Disease Prevention and Control [ECDC], 2017). Seventy-nine C. coli isolates (59.4%) and three C. jejuni isolates (8.5%) showed resistance to ciprofloxacin and erythromycin (treatment used against campylobacteriosis before onset of resistance and the current treatment against this bacteria). Campylobacter isolates resistant to erythromycin (Table 1) were analyzed for the presence of RNA methylase gene erm(B).

erm(B) Allelic Variation among Bacterial Genera, Hosts, and Countries

Comparison of sequence homology can provide information about the horizontal transfer of resistance genes, including erm(B), among bacterial species. The nucleotide sequences of 10 erm(B) genes present in Campylobacter were compared, two from this study and eight of Chinese and Spanish origin. Using the first erm(B) sequence described in Campylobacter as reference (C. coli ZC113; GenBank accession number: KC575115), four alleles have been identified in the erm(B) sequences from Campylobacter (Supplementary Table S3). Allele 1 was the most common (7/10) and was used as reference. Alleles 2 and 3 were present in only one C. coli isolate each from Spain, with SNPs A299G (Asn-100-Ser) and A353G (His-118-Arg), respectively. Allele 4 was found in a C. jejuni isolate from China and is characterized by the synonymous SNP C726T. Bacterial genera, hosts and origins with erm(B) sequence identical to the four alleles observed in Campylobacter were identified in the NCBI database, compiled (Supplementary Table S3) and compared (Figure 2). The erm(B) alleles detected in Campylobacter had been previously identified mainly in Enterococcus and Staphylococcus isolated from humans and pigs in Asia and Europe. Allele 1 of erm(B) is represented in 31 sequences from eight bacterial species, including Streptococcus suis (11/31; 35.5%), Enterococcus faecium (7/31; 22.5%), and C. coli (7/31; 22,5%). S. suis isolates were mainly isolated in China (7/11; 63.6%) and all were from swine hosts. Sequences of E. faecium were mainly from Japan (4/7; 57.1%) and all were of human origin. The majority of erm(B) allele 1 sequences from Campylobacter were of Chinese origin (6/7; 85.7%) and sampled from humans, swine, and chickens. Allele 2 of erm(B) was detected in 16 bacterial species mainly Clostridium difficile (10/39; 25.6%) and E. faecium (7/39; 17.9%), from European and United States people and pigs of Chinese origin, respectively. Allele 2 of erm(B) is slightly less common in NCBI database than allele 1, being the only one identified in six bacterial genomes, mainly E. faecium (10/22; 45.4%) sampled from humans in the United States, Australia, Japan and South Korea, whereas allele 4 of erm(B) was not identified in any other sequence available in public databases.

FIGURE 2

FIGURE 2. Host and geographical distribution of the erm(B) alleles identified in Campylobacter in this study among other bacterial genera. Allele 1 belongs to C. coli ZTA14/01426 (MF134832), allele 2 belongs to C. coli ZTA09/02204 (KT953380), allele 3 belongs to C. coli ZTA14/01086 (MF134831) and allele 4 belongs to C. jejuni C179b (KF864551). erm(B) homologs were identified in GenBank using BLAST with a coverage and similarity of 100%. Accession numbers of each sequence is given in Supplementary Table S3. Sequences without host data and geographical location have not been included. Exact identity of erm(B) alleles between species, host/environment and origin of isolation is represented by a connection (width proportional to relative prevalence).

Discussion

Definitive characterization of erythromycin resistance in bacterial pathogens is an important objective defined by the EFSA and the European Centre for Disease Prevention and Control (ECDC) (European Food Safety Authority [EFSA]/European Centre for Disease Prevention and Control [ECDC], 2017). To date, the erm(B) gene has been identified in Campylobacter isolates from swine, chickens, ducks and humans from China and in one broiler sample from Spain (Wang et al., 2014; Florez-Cuadrado et al., 2016). Monitoring antimicrobial resistance in Campylobacter isolated from turkeys has been mandatory since 2014 in European countries where the production of turkey meat exceeds 10,000 tons per year (2016). The occurrence of erythromycin resistant C. coli isolates from turkeys in Germany, Romania and Spain had risen to 43.3% in 2014 (2016), compared to previous surveys where lower frequencies were detected for poultry (14.5%) and swine (20.7%) (European Food Safety Authority [EFSA]/European Centre for Disease Prevention and Control [ECDC], 2015, 2016b). Since Campylobacter infections are related to consumption of food from animal origin, these levels of resistance could potentially produce therapeutic failure of antibiotic treatment for campylobacteriosis in humans.

Increased antimicrobial resistance among Campylobacter populations is a consequence of the widespread acquisition of antimicrobial resistance and clonal expansion of resistant lineages (Wimalarathna et al., 2013). Campylobacter can acquire DNA, including antimicrobial resistance genes, from relatively distantly related lineages through HGT, involving the replacement of homologous sequences, or the acquisition of mobile genetic elements (MGEs). There is evidence that plasmid acquisition mediates Campylobacter resistance to tetracycline, chloramphenicol and aminoglycosides (Courvalin et al., 1978; Taylor et al., 1987; Wang and Taylor, 1990) but in some cases, these resistances might be conferred by polymorphism of chromosomal sequences. This is also the case in Campylobacter for resistance to fluoroquinolones and macrolides, mediated by mutations in gyrA or 23S rDNA sequences, respectively (Engberg et al., 2001). Mutations that confer antimicrobial resistance can occur independently in multiple lineages but can also spread by natural transformation followed by homologous recombination, leading to the dissemination of antimicrobial resistance among bacteria that share an ecological niche (Meric et al., 2015).

Although the presence of the erm(B) gene was evaluated on a total of 85 Campylobacter isolates, the scope of the genomic comparison was limited to only two C. coli because they were positive for the erythromycin resistance gene. Despite the limited number of genomes sequenced, the erm(B)-carrying genomic islands identified in Campylobacter isolated from Spain show genetic differences in comparison with ones from China. Thus, the genomic islands identified in Spain do not correspond to any of the six types of genomic islands identified in C. coli from China (Qin et al., 2014; Wang et al., 2014; Florez-Cuadrado et al., 2016). All of the erm(B)-carrying genomic islands posses aminoglycoside resistance genes but the gentamicin resistance gene aph(2”)-IIIa is present only in Spanish isolates. Resistance to gentamicin was represented in the Chinese erm(B)-carrying sequences with the presence of the aacA-aphD gene and was identified in C. coli isolates from humans, poultry, and swine (Wang et al., 2014).

Allele 2 of the erm(B) gene has been identified in more bacterial genera than the other alleles in this study (Figure 2). All bacterial genera where erm(B) allele 2 has been identified belong to the Firmicutes, with two exceptions that belong to the Actinobacteria. These bacterial genera are present in the gastrointestinal tract and species like Enterococcus, Streptococcus, and Staphylococcus share the niche with Campylobacter (Zilhao et al., 1988). The transfer of antibiotic resistance genes in the human colon between bacteria of different genera has been reported (Huddleston, 2014) and it is possible that the antibiotics supplied in both animals and humans could facilitate the selection of strains carrying these multiresistant genomic islands and favor their dispersion. Identifying erm(B) genes and MDR genomic islands in Campylobacter isolates from turkeys, adds another component to the already extensive network of bacterial genera and hosts involved in the possible dispersion of critical antibiotic resistance genes and MGEs. This study highlights the need to sequence a greater number of Campylobacter genomes with the objective of evaluating the impact of genomic islands on the dispersion of antimicrobial resistance genes in this genus.

Funding

This work was partially supported by the Ministry of Science and Innovation (AGL2009-07550; AGL2012-39028), Ministry of Agriculture, Food and Environment (2014/000223), Autonomous Community of Madrid, Spain (S2009/AGR-1489; S2013/ABI-2747), and by the Spanish Ministry of Economy and Competitiveness (AGL2012-39028). DF-C is supported by the FPI program (BES-2013-065003) from the Spanish Ministry of Economy and Competitiveness. SS is funded by grants from the Medical Research Council (MR/L015080/1).

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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